Objective A rapid method based on matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS) for the identification of intact bacterial cells was used for the discrimination of Salmonella strains at genus and species level. The aim of this study was to discuss the relationship of the result from MALDI-TOF-MS analysis and serotyping, and the typing ability of MALDI-TOF-MS to Salmonella strains through relative soft wares. Method Twenty-four Salmonella strains isolated from the Foodborne Disease Monitoring System of Liaoning Province in 2008 and 2 standard strains purchased from ATCC were used in the study. Sixteen Salmonella strains belonging to four serotypes were applied on MALDI-TOF-MS for the identification of Salmonella at species level. A detailed analysis on mass spectrometric profiles and the information of protein peaks were obtained from four different Salmonella species. The homology of these 4 Salmonella enteritidis was also discussed through biochemical test and relative analysis with MALDI-TOF-MS. After an optimum medium suitable for both the growth of Salmonella strains and MALDI-TOF-MS analysis was selected, 26 Salmonella strains were tested to verify the accuracy of MALDI-TOF-MS in genus levels, and 16 of 26 Salmonella species, which belonging to 4 different serotypes, were also typed in species level. Relative software was used to collect data and compare the peaks obtained from four Salmonella species. The biochemical test, MALDI-TOF-MS dendrogram analysis and Principal Component analysis were adopted to confirm the homologous diversity of 4 Salmonella enteritidis from different locations. Results The identification results of MALDI-TOF-MS on 26 Salmonella in genus level were matched with their classic biochemical identification results, and the discrimination results of 16 Salmonella in species level were also matched with their serotypes. Slight differences among four Salmonella species were also discovered through the peaks lists of them by MALDI-TOF-MS successfully. Furthermore, the homologous diversity of 4 Salmonella enteritidis was demonstrated obviously. Conclusion MALDI-TOF-MS can be used to discriminate Salmonella strains used in this study rapidly and exactly on genus level. It has also a potential ability on typing Salmonella species and will be a powerful tool for rapid and accurate identification of foodborne pathogens.